Polypeptide for the treatment of cancer and a method for preparation thereof

ABSTRACT

This invention characterizes the specific peptide fragment derived from specially prepared zinc charged fetuin and a method of preparation thereof, wherein the fragment was found to contain an apoptosis-inducing activity. Specifically, the amino acid sequence of this peptide is His-Thr-Phe-Met-Gy-Val-Val-Ser-Leu-Gly (SEQ ID NO: 3) and correlates to amino acid no. 300-309 of bovine fetuin, referred to herein as Fetuin Peptide Fragment (FPF 300-09). FPF 300-09 strongly induced apoptosis in LNCaP (prostate cancer) and HT-29 (colon cancer) cells without affecting CCD 18 Co (normal colon) cells. The in vitro tissue culture study demonstrated that the FPF 300-09 is more potent than the parent molecule (full-length zinc charged fetuin) in inducing apoptosis. FPF 300-09 has a LD 50  of 0.3-0.4 μM, while the LD 50  for zinc-charged fetuin is 3-10 μM.

CLAIM OF PRIORITY

This application is a divisional application from a currently U.S.application Ser. No. 10/772,537, filed Feb. 5, 2004 (now a U.S. Pat. No.7,193,040), which is a divisional application from the U.S. applicationSer. No. 10/145,682, filed May 14, 2002 (now a U.S. Pat. No. 6,720,311,which is a continuation-in-part to the U.S. application Ser. No.09/902,208, filed Jul. 9, 2001 (now a U.S. Pat. No. 6,737,402), which isa continuation-in-part to the U.S. application Ser. No. 09/414,136,filed Oct. 7, 1999 (now a U.S. Pat. No. 6,258,779), which is acontinuation-in-part to the U.S. application Ser. No. 09/149,878, filedSep. 8, 1998 (now a U.S. Pat. No. 5,994,298), which is acontinuation-in-part to the U.S. application Ser. No. 08/993,432, filedDec. 18, 1997, now abandoned.

BACKGROUND OF THE INVENTION

Human beings have had a long battle against cancer. Because the diseaseis so widespread, manifests itself in so many different ways and is sorelentless, the potential market for effective cancer therapies isenormous. It is estimated that 10 million people in the U.S. either haveor have had cancer. The National Cancer Institute (NCI) projects that in1995, some 1.2 million new cases of cancer will be diagnosed in theUnited States, and that 538,000 people will die of the disease.

Cancer is currently treated, with a low degree of success, withcombinations of surgery, chemotherapy and radiation. The reason of thelow degrees of success in cancer chemotherapy is as the following:current chemotherapeutic approaches target rapidly dividing tumor cells.This approach is ineffective when the cancer is dormant or growingslowly. Such treatments also affect other, non-cancerous cells thatdivide rapidly, causing harmful side effects.

Only in the last several years has a new approach emerged in the battleagainst cancer. This approach is based on the newly discoveredbiological phenomenon called “Apoptosis”. Apoptosis is also called“programmed cell death” or “cell suicide”. (Krammer, et al., “Apoptosisin the APO-1 System”, Apoptosis: The molecular Basis of Cell Death, pp.87-99 Cold Spring Harbor Laboratory Press, 1991). In contrast to thecell death caused by cell injury, apoptosis is an active process ofgene-directed, cellular self-destruction and that it serves abiologically meaningful function. (Kerr, J. F. R and J. Searle J.Pathol. 107:41, 1971).

One of the examples of the biologically meaningful functions ofapoptosis is the morphogenesis of embryo. (Michaelson, J. Biol. Rev.62:115, 1987). Just like the sculpturing of a sculpture, which needs theaddition as well as removal of clay, the organ formation (Morphogenesis)of an embryo relies on cell growth (addition of clay) as well as celldeath (removal of clay). As a matter of fact, apoptosis plays a key rolein the human body from the early stages of embryonic development throughto the inevitable decline associated with old age. (Wyllie, A. H. Int.Rev. Cytol. 68:251, 1980). The normal function of the immune,gastrointestinal and hematopoietic system relies on the normal functionof apoptosis. When the normal function of apoptosis goes awry, the causeor the result can be one of a number of diseases, including: cancer,viral infections, auto-immune disease/allergies, neuro-degeneration orcardiovascular diseases.

Because of the versatility of apoptosis involved in human diseases,apoptosis is becoming a prominent buzzword in the pharmaceuticalresearch field. Huge amounts of time and money are being spent in anattempt to understand how it works, how it can be encouraged orinhibited and what this means for practical medicine. A handful ofcompanies have been formed with the prime direction of turning work inthis nascent field into marketable pharmaceutical products. Theemergence of a core of innovative young companies combined with thetentative steps being taken by established industrial players arecertain to make apoptosis research one of the fastest-growing and mostpromising areas of medical study of the 1990's.

The idea that cancer may be caused by insufficient apoptosis merged onlyrecently (Cope, F. O. and Wille, J. J., “Apoptosis”: The Molecular Basisof Cell Death, Cold Spring Harbor Laboratory Press, p. 61, 1991). Thisidea however, opens a door for a new concept in cancer therapy—cancercells may be killed by encouraging apoptosis. Apoptosis modulation,based on the processes present in normal development, is a potentialmechanism for controlling the growth of tumor cells. Restoring apoptosisin tumor cells is an attractive approach because, at least in theory, itwould teach the cells to commit suicide.

Nevertheless, since the objective of cancer treatment is to kill cancercells without killing the host, although apoptosis may open a new doorfor cancer therapy by inducing apoptosis in tumor cells, the success ofthis treatment is still dependent on the availability of drugs that canselectively induce apoptosis in tumor cells without affecting normalcells. In this patent application, we describe the methods for theisolation of proteins that specifically induce apoptosis in cancer cellswithout effect in normal cells. These proteins may present a new classof anticancer drugs that induce apoptosis in cancer cells, which mayoffer a breakthrough in cancer therapy.

SUMMARY OF THE INVENTION

The purpose of this invention is to characterize the specific peptidefragment derived from specially prepared zinc charged fetuin wherein thefragment was found to contain an apoptosis-inducing activity.Specifically, the amino acid sequence of this peptide is His Thr Phe SerGly Val Ala Ser Val Glu (SEQ ID NO:1) and correlates to amino acid no.300-309 of fetuin, referred to herein as Fetuin Peptide Fragment (FPF300-09). FPF 300-09 strongly induced apoptosis in LNCaP (prostatecancer) and HT-29 (colon cancer) cells without affecting CCD 18 Co(normal colon) cells. The in vitro tissue culture study demonstratedthat the FPF 300-09 is more potent than the parent molecule in inducingapoptosis. FPF 300-09 has a LD₅₀ of 0.3-0.4 μM, while the LD₅₀ forzinc-charged fetuin is 3-10 μM.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a table of test data of using fetal fetuin on mice bearingleukemia.

FIG. 2 shows a table of the comparison of original fetuin with zinc incomparison with the supercharged zinc fetuin to reach LD₅₀.

FIG. 3 shows another table of the comparison of original fetuin withzinc in comparison with the supercharged zinc fetuin to reach LD₅₀.

FIG. 4 shows a slide of LNCaP (prostate cancer) cells without treatmentof filtrate containing FPF 300-09.

FIG. 5 shows a slide of LNCaP cells incubated with filtrate containingFPF 300-09 for six (6) hours.

FIG. 6 shows an additional slide of LNCaP (prostate cancer) cellswithout treatment of filtrate containing FPF 300-09.

FIG. 7 shows a slide of LNCaP cells incubated with filtrate containingFPF 300-09 and which expressed membrane blebbing, which is an indicatorof cells undergoing apoptosis.

FIG. 8 shows a table illustrating, among other things, the effect ofincubating the filtrate containing FPF 300-09 treated with proteinase K.

FIG. 9 shows a slide of LNCaP cells without filtrate containing FPF300-09.

FIG. 10 shows a slide of LNCaP cells, which were incubated with filtratecontaining FPF 300-09 for three (3) hours and expressed membrane“blebbing,” which is an indicator of cells undergoing apoptosis.

FIG. 11 is a graph of FPF 300-09 (nM) versus the percent apoptosis inHT-29, CCD-18 Co, and LNCaP cells.

FIG. 12 is a graph of the time of incubation of FPF 300-09 filtrateversus percent apoptosis in HT-29 and CCD-18 Co cells.

FIG. 13 is a table of the LD₅₀ values for fetuin and various fetuinfragments, including FPF 300-09.

DETAILED DESCRIPTION OF THE INVENTION

This patent application describes the isolation of five proteins: ApogenP-1a, Apogen 1b, Apogen 1c, Apogen P-2 and Apogen L.

(A) Isolation of Apogen P-1

(1) Source of Apogen P-1

Apogen P-1 was isolated from the conditioned medium of a cell linecalled XC, which was derived from rat tumor (ATCC CCL 165). XC cellswere first grown in Dulbecco's Modification of Eagle's Medium (DMEM)containing 10 % Fetal bovine serum (FBS) for 3 days. XC cells were thenwashed with PBS (3×100 ml) to remove serum and then grown in DMEMcontaining no FBS for 4 days. From this serum free conditioned medium,we detected an activity inducing apoptosis in a prostate cancer cellline called LNCAP. On the other hand, normal human lung fibroblast cellline (CCD 39 Lu) and breast cancer cells (MCF-7) is not affected by thisactivity.

(2) Activity of Apogen P-1

(a) Apoptosis Inducing Activity

The activity of the crude conditioned medium of XC cells was tested onthe following cell lines: JEG-3 (Choriocarcinoma), G401 (Wilm's tumor)LNCAP (Prostate cancer), T84 (colon cancer), HL-60 (leukemia), breastcancer cells (MCF-7), -and CCD 39 Lu (normal lung fibroblast). When 10folds concentrated conditioned medium was incubated for 18 hours withthe above cell lines in the presence of 5% serum, the conditioned mediuminduced apoptosis in JEG-3 cells (35%), G 401 cell (27%), LNCaP(100 %)and without activity in CCD 39 Lu (0%), T84(0%), MCF-7(0%) andHL-60(0%).

Apoptosis is a distinct type of cell death that differs fundamentallyfrom degenerative death or necrosis in its nature and biologicalsignificance. A cell undergoing apoptosis is distinct from a cellundergoing necrosis both morphologically and biochemically.Morphologically, the earliest definitive changes in apoptosis that havebeen detected with the electron microscope are compaction of the nuclearchromatin into sharply circumscribed, uniformly dense masses about thenuclear envelope and condensation of the cytoplasms. Phase-contrastmicroscope of cells under apoptosis shows the condensation and thefragmentation of DNA and the budding of cell to form apoptotic body.

To morphologically demonstrate that the XC conditioned medium containsactivity inducing apoptosis, LNCAP cells were incubated with controlmedium or the conditioned medium treated as described as above for 15 hrand then stained with Hoechst dye for 2 hours. The nuclei of the LNCAPcells that have been incubated with the control medium are normal andhealthy. However, the nuclei of the LNCAP cells that have been incubatedwith the conditioned medium (X20, exchanged to RPMI) show thecharacteristics of apoptosis. First, the conditioned medium causes thecondensation of the nucleus, demonstrated by the more intensefluorescent light compared with the control nucleus. Secondly, thenucleus condensation is accompanied by the fragmentation of DNA,demonstrated by the breakage of the nucleus. As we have mentioned above,the condensation of the nucleus and the DNA fragmentation are themorphological characteristics of cells under apoptosis. These resultssuggest that the conditioned medium from XC cells contain an activityinducing apoptosis in LNCAP cells. On the other hand, the conditionedmedium fails to induce apoptosis in normal human lung fibroblast (CCD 39Lu cells) and breast cancer cells (MCF-7). The nuclei of CCD 39 Lu cellsremain the same with or without incubating with the conditioned mediumof XC cells.

(b) Cell Repelling Activity

The partially purified Apogen P-1b (Q2 anionic exchanger chromatographystep) isolated as described below was recently found to contain anactivity other than inducing apoptosis. We found that Apogen P-1b havethe activity to repel cells away. This activity is opposite to that ofgrowth factors; many growth factors such as Platelet Derived GrowthFactor (PDGF), Epidermal Growth factor (EGF), Fibroblast Growth factor(FGF) or Transforming Growth factor (TGF) function as a“chemoattractant”—which means that these growth factors attract cellstoward them. (Grotendorst, G. R. et al., Proc. Natl. Acad. Sci. 78:3669,1981; Grant, M. B. et al Invest. Ophthal. Visual Science. 33:3292,1992). This finding suggests that Apogen P-1b isolated in this inventionplays an opposite biological function as that of a growth factor. Forexample, growth factors induce cell growth and attract cells, whereasApogen P-1b induces cell death and repels cells. Apogen P-1b is thefirst “chemorepellent” found in the field of modern biology.

A tissue culture device called Transwell Insert purchased from Costar(Cambridge, Mass.) was used to discover the chemorepellent activity ofApogen P-1b. This device, which has been widely used for the studies ofcell migration/invasion, contains an upper chamber and a lower chamber.Between these two chambers is a polyester microporous membrane with 3.0μm pore size, which allows cells to migrate through the membrane. Testedcells are grown on the upper chamber, and the tested compound is placedin the lower chamber. If this tested compound is a chemo-attractant, weshould see more cells migrate through the membrane than the controlsample. In our experiments, Hep G2 (100,000 cells) cells, which have acell size 3-4 times as big as the membrane pore size, were grown in theupper chamber for 2 hours, and then, the partially purified Apogen-1b(30 μl) isolated by ammonium sulfate precipitation and Q2 HPLCchromatography as described above was placed in the lower chamber. After15 hours, cells that have migrated through the membrane were collectedby treating the membrane with 0.2 ml of trypsin solution for 30 min.Cells in ten micro-liters of the trypsin solution were counted in ahemacytometer.

In several experiments, we found that the partially purified Apogen-1bcontained an activity decreasing the number of cells going through themembrane. For example, in one experiment, in the presence of thepartially purified Apogen P-1b, the cell number in 10 micro-literstrypsin solution (which are the cells go through membrane) is 24+−4,whereas the number of cells that go through the membrane in the controlexperiment is 82+−27. This result suggests that the partially purifiedApogen P-1b prevents Hep G2 cells from migrating through membrane.

To unequivocally show that Apogen P-1b repel cells, an invertedexperiment was installed, instead of placing Apogen P-1b in the lowerchamber, we placed Apogen P-1b in the upper chamber, after 12 hours, wefound that 56+−19 cells went through membrane compared with controlexperiment of 30+−1.7 cells per 10 micro-liters of trypsin solution. Thestatistically significant increase or decrease in the number of cellsgoing through the membrane by alternatively placing Apogen P-1b in theupper or lower chamber of this tissue culture device strongly suggeststhat Apogen P-1b repels cells.

(3) Isolation of Apogen P-1 from XC Conditioned Medium

The Apogen P-1 present in the conditioned medium was isolated by thefollowing steps:

Step 1: Ammonium Sulfate Precipitation

Apogen P-1 was precipitated by 80% saturated of ammonium sulfate byadding 561 g of ammonium sulfate per liter of conditioned medium. Pelletwas collected by centrifugation and the proteins were dissolved in 10 mMTris-HCl (pH 7.4). After removal of ammonium sulfate by dialysis, thedissolved proteins were separated by a Q2 HPLC column.

Step 2: Q2 HPLC Chromatography

The dissolved proteins isolated by ammonium sulfate precipitation wereconcentrated and loaded on to a Q2 column (Bio-Rad) which was furtherdeveloped by a linear gradient constructed by buffer A (10 mM Tris-HCl,pH 7.4) and buffer B (10 mM Tris-HCl, pH 7.4, 0.55 M NaCl) usingBioRad's BioLogic HPLC system. The linear gradient was constructed byincreasing buffer B from 0% to 100% in buffer A within 10 min (20milliliter elution volume and thereafter the column was eluted with 100%buffer B for 5 min.

The Apogen P-1 activity was assayed by the induction of apoptosis inLNCAP cells. We found that there were three activity peaks across thechromatogram profile. Fraction 5 to 7 caused 70% cell death; fraction8-10 caused 65% cell death; and fraction 11-14 caused 90% cell death in18 hr. We collected fractions 5-7 and named it Apogen P-1a; fractions8-10 were named Apogen P-1b; and fractions 11 to 14 were named ApogenP-1c. These three Apogen P-1's were further purified by a reverse phasecolumn.

Step 3: Reverse Phase Chromatography.

Apogen P-1a, Apogen P-1b and Apogen P-1c were separately concentrated to1.5 ml. One ml of methanol containing 0.05% trifluoracetic acid wasadded. In each sample, a large amount of protein was precipitated bythis treatment, whereas the apoptosis inducing activity remained in thesupernatant. The supernatant was then applied to a reverse phase RP-4column (Micra Scientific Inc) and developed by a linear gradientconstructed by solution A (H20, 0.05% TFA) and solution B (Methanol,0.05% TFA). The linear gradient was constructed by increasing solution Bfrom 0% to 100% in solution A within 10 min (20 milliliter elutionvolume and thereafter the column was eluted with 100% solution B for 5min).

Step 4: Preparative Electrophoresis.

Apogen 1c isolated by anion exchange chromatography was purified by bothreverse phase chromatography (step 3) and preparative electrophoresis bya MiniPrep Gel electrophoresis (Bio-Rad). In the reverse phasechromatogram of Apogen P-1a, fractions 12-13 had activity inducing 80%cell death in LNCAP cells at 10 hr. In the reverse phase chromatogram ofApogen P-1b, fractions 14 and 15 had activity inducing 45% cell death inLNCAP cells at 18 hr.

In the reverse phase chromatogram of Apogen P-1c, fraction No. 5 hadactivity inducing 52% cell death in LNCAP cells at 18 hr.

The purity of the isolated Apogen P-1a, Apogen P-1b and Apogen P-1c waschecked with SDS-polyacrylamide gel electrophoresis stained with silverstaining.

-   (1) Apogen P-1a: a protein band with molecular weight of 70 KD was    obtained. This result suggests the nearly successful purification of    Apogen P-1a, which has the molecular weight of 70 KD on SDS-PAGE.-   (2) Apogen P-1b: A single faint protein band with molecular weight    of 55 KD was obtained. This result suggests the successful    purification of Apogen P-1b, which has the molecular weight of 55 KD    on SDS-PAGE.-   (3) Apogen P-1c: The purification of Apogen 1c by Reverse Phase    chromatography leads to the isolation of a 70 KD protein whereas the    purification of Apogen-1c by preparative electrophoresis leads to    the purification of a 57 KD protein. A major protein band with    molecular weight of 70 KD was obtained by Reverse Phase    chromatography. A 57 KD protein, on the other hand, was isolated by    preparative electrophoresis.

Our next step, obviously, will be put our entire efforts on obtainingenough of the protein band for an amino acid sequence.

(B) Isolation of Apogen P-2

(1) Source of Apogen P-2

Apogen P-2 was isolated from the conditioned medium of a cell linecalled C3H 1OT1/2, which was derived from mouse embryo cells (ATCC CCL226). C3H 1OT1/2 cells were first grown in alpha Modification of Eagle'sMedium (alpha-MEM) containing 10% Fetal bovine serum (FBS) for 3 days.Cells were then washed with PBS (3×100 ml) to remove serum and thengrown in alpha-MEM containing no FBS for 4 days. From this serum freeconditioned medium, we detected an activity inducing apoptosis in aprostate cancer cell line called LNCAP. On the other hand, normal humanlung fibroblast cell line (CCD 39 Lu) was not affected by this activity.

(2) Activity of Apogen P-2

(a) Apoptosis Inducing Activity

The activity of the crude conditioned medium of C3H 1OT1/2 cells wastested on the following cell lines: LNCAP (Prostate cancer), breastcancer cells (MCF-7), and CCD 39 Lu (normal lung fibroblast). When the10-fold concentrated conditioned medium was incubated for 18 hours withthe above cell lines in the presence of 5% serum, the conditioned mediuminduced apoptosis in LNCaP(100%) and without activity in CCD 39 Lu (0%).To morphologically demonstrate that the C3H 10TI/2 conditioned mediumcontains activity inducing apoptosis, LNCAP cells were incubated withcontrol medium or with the conditioned medium treated as described asabove for 15 hr and then stained with Hoechst dye for 2 hours. Thenuclei of the LNCAP cells that have been incubated with control mediumare normal and healthy. However, the nuclei of the LNCAP cells that havebeen incubated with the conditioned medium show the characteristic ofapoptosis. First, the conditioned medium causes the condensation of thenucleus, demonstrated by the more intense fluorescent light as comparedwith the control nucleus. Secondly, the condensation of the nucleus isaccompanied by the fragmentation of DNA, demonstrated by the breakage ofnucleus.

As we have mentioned above, the condensation of the nucleus and DNAfragmentation are the morphological characteristics of cells underapoptosis. The same holds true of breast cancer cells (MCF-7) in which85% apoptotic effect was observed after 18 hours of exposure to P-2.These results suggest that the conditioned medium from C3H1OT1/2 cellscontains an activity inducing apoptosis in LNCAP and MCF-7 cells. On theother hand, the conditioned medium fails to induce apoptosis in normalhuman lung fibroblast (CCD 39 Lu cells). The nuclei of CCD 39 Lu cellsremain the same with or without incubating with the conditioned mediumof C3H1OT1/2 cells.

(b) Cell Repelling Activity

The partially purified Apogen P-2, isolated by ammonium sulfateprecipitation, hydroxylapatite, and heparin treatment as describedabove, was recently found to contain an activity other than inducingapoptosis. Similar to Apogen P-1b, Apogen P-2 has the activity to repelcells away. Transwell Insert purchased from Costar (Cambridge, Mass.)was used to discover the chemorepellent activity of Apogen P-2. Thisdevice, which has been widely used for the study of cellmigration/invasion, contains an upper chamber and a lower chamber.Between these two chambers is a polyester microporous membrane with 3.0μm pore size, which allows the cells to migrate through the membrane.The tested cells (HL-60) were grown on the upper chamber, and the testedcompound (Apogen P-2) was placed in the lower chamber.

In our experiments, HL-60 (100,000 cells) cells, which have a cell size2-3 times as big as the membrane pore size, were grown in the upperchamber for 2 hours, and then, the partially purified Apogen P-2 (30 μl)isolated by ammonium sulfate precipitation, hydroxylapatite, and Heparinagarose as described above was placed in the lower chamber. After 6hours, cells that have migrated through the membrane were collected fromthe lower chamber; the medium in lower chamber (0.6 ml) was centrifugedfor 10 min; and the HL-60 cells that went through the membrane werecollected and re-suspended in 80 μl of PBS. The cells in tenmicro-liters of the PBS solution were counted in a hemacytometer.

In several experiments, we found that the partially purified Apogen P-2contained an activity that decreases the number of cells going throughthe membrane. For example, in one experiment, in the presence of thepartially purified Apogen P-2, the number of cells in 10 micro-litersPBS solution (which are the cells go through the membrane) is 47+−5.6,whereas the number of cells that go through the membrane in the controlexperiment is 213+−40. At this moment, no apoptosis was observed inHL-60 cells present in the upper chamber. This result suggests that thepartially purified Apogen P-2 prevents the HL-60 cells from migratingthrough membrane.

(3) Isolation of Apogen P-2 from C3H1OT1/2 Conditioned Medium

The Apogen P-2 present in the conditioned medium was isolated by thefollowing steps:

Step 1: Ammonium Sulfate Precipitation.

Apogen P-2 was precipitated by ammonium sulfate (80% saturated) byadding 561 g of ammonium sulfate per liter of conditioned medium. Thepellet was collected by centrifugation, and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4).

Step 2: Hydroxylapatite Treatment.

After removal of ammonium sulfate by dialysis in 10 mM Tris-HCl (pH7.5), the dissolved proteins were incubated with Hydroxylapatite gel(Bio-Gel HTP gel, Bio-Rad) for 1 hr. After removing HTP gel bycentrifugation, the activity inducing apoptosis in LNCAP cells was foundto be present in the supernatant, which was then further treated withHeparin agarose gel.

Step 3: Heparin Agarose Treatment.

The supernatant from step 2 was further incubated with Heparin agarose(Sigma) for 1 Hr. After removing HTP gel by centrifugation, the activityinducing apoptosis in LNCAP cells was found to be present in thesupernatant.

Step 4: Reverse Phase Chromatography.

Apogen P-2 present in the supernatant of Heparin agarose in step 3 wasfurther purified by reverse phase chromatography. Apogen P-2 wasconcentrated to 1 ml. One milliliter of methanol containing 0.05%Trifluoracetic acid was added. A large amount of protein wasprecipitated by this treatment, whereas the apoptosis inducing activity(P-2) remained in the supernatant. The supernatant was then applied to areverse phase RP-4 column (Micra Scientific Inc) and developed by alinear gradient constructed by solution A (H20, 0.05% TFA) and solutionB (Methanol, 0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A in 10 min (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min.

In the reverse phase chromatogram of Apogen P-2, fractions 12-14 haveactivity inducing 80% cell death in LNCAP cells at 12 hr. The purity ofthe isolated Apogen P-2 was checked with SDS-polyacrylamide gelelectrophoresis stained with silver staining, and a single protein bandwith molecular weight of 65 Kd was obtained.

(C) Isolation of Apogen L

(1) Source of Apogen L

Apogen L was isolated from the conditioned medium of XC cell line (ATCCCCL 165). XC cells were grown in Dulbecco's Modification of Eagle'sMedium (DMEM) containing 10% Fetal bovine serum (FBS) for 4 days. Fromthis conditioned medium, we detected an activity inducing apoptosis in aleukemia cell line called HL-60. On the other hand, normal human lungfibroblast cell line (CCD 39 Lu) is not affected by this activity.

(2) Isolation of Apogen L from XC Conditioned Medium

The Apogen L present in the conditioned medium was isolated by thefollowing steps:

Step 1: DE52 Absorption

The conditioned medium was incubated with the anion exchanger, DE 52(Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubation mixturewas centrifuged and DE 52, which binds Apogen L was collected and washedwith 10 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. Apogen L was theneluted from DE 52 cellulose by 10 mM Tris-HCl (pH 7.5) containing 0.5 MNaCl.

Step 2: Heparin Agarose Absorption

Apogen L isolated as described in step 1 was further absorbed by Heparinagarose (Sigma) by incubating Apogen L with Heparin agarose for 1 hr.Heparin agarose was collected by centrifugation and was washed with 10mM Tris-HCl (pH 7.5). Apogen L absorbed in Heparin agarose was theneluted by 2 M NaCl.

Step 3: Q2 HPLC Chromatography

Apogen L isolated as described above was concentrated and loaded onto aQ2 column (Bio Rad) which is further developed by a linear gradientconstructed by buffer A (10 mM Tris-HCl, pH 7.4) and buffer B (10 mMTris-HCl, pH 7.4, 0.5 M NaCl) using Bio-Rad's BioLogic HPLC system. Thelinear gradient was constructed by increasing buffer B from 0% to 100%in buffer A within 10 min. The purity of the isolated Apogen L waschecked with SDS-polyacrylamide gel electrophoresis stained with silverstaining. A single protein band with molecular weight of 55 Kd wasobtained.

(3) Activity of Apogen L

The activity of Apogen L isolated as described above was tested on thefollowing cell lines: HL-60 (leukemia) and CCD 39 Lu (normal lungfibroblast). To morphologically demonstrate that Apogen L containsactivity-inducing apoptosis, HL-60 cells were incubated with Apogen Lisolated as described as above for 15 hr and then stained with Hoechstdye for 2 hours. The nuclei of the HL-60 cells that have been incubatedwith control medium are normal and healthy.

However, the nuclei of the HL-60 cells that have been incubated withApogen L show the characteristic of apoptosis. First, Apogen L causesthe condensation of nucleus, demonstrated by the more intensefluorescent light compared with the control nucleus. Secondly, thenucleus condensation is accompanied by the fragmentation of DNA,demonstrated by the breakage of nucleus. As we have mentioned above, thenucleus condensation and DNA fragmentation are the two morphologicalcharacteristics of cells under apoptosis. These results suggest that theisolated Apogen L contains an activity inducing apoptosis in HL-60cells. Apogen L also induces apoptosis in MCF-7 (breast cancer) cells.On the other hand, the conditioned medium fails to induce apoptosis innormal human lung fibroblast (CCD 39 Lu cells).

EXAMPLES

A. Methods

1. Preparation of Condition Media.

A. Preparation of XC Condition Medium for Isolation of Apogen P-1.

Apogen P-1 was isolated from the conditioned medium of a cell linecalled XC, which was derived from a rat tumor (ATCC CCL 165). XC cellswere first seeded in a roller bottle (Polystyrene, area surface=850 Cm2,Corning) in Dulbecco's Modification of Eagle's Medium (DMEM) containingCO2, 10% fetal bovine serum (FBS), non-essential amino acids, penicillinand streptomycin for 3 days. XC cells were then washed with PBS (3×100ml) to remove serum and then grown in 100 ml of DMEM containing no FBS(with CO2), non-essential amino acids, penicillin and streptomycin) for4 days. The conditioned medium was collected and clarified bycentrifugation.

B. Preparation of C3H 1OT1/2 Condition Medium for Isolation of ApogenP-2.

Apogen P-2 was isolated from the conditioned medium of a cell linecalled C3H1OT1/2, which was derived from a mouse embryo and waspurchased from American Type Culture Collection (ATCC CCL 226).C3H1OT1/2 cells were first seeded in a roller bottle (Polystyrene, areasurface=850 Cm2, Corning) in alpha Modification of Eagle's Medium(alpha-MEM) containing CO2, 10% Fetal bovine serum (FBS), penicillin andstreptomycin for 3 days. C3H1OT1/2 cells were then washed with PBS(3×100 ml) to remove serum and then grown in 100 ml of alpha MEMcontaining no FBS (with C02, penicillin and streptomycin) for 4 days.The conditioned medium was collected and clarified by centrifugation.

C. Preparation of XC Condition Medium for Isolation of Apogen L.

Apogen L was isolated from the conditioned medium of a cell line calledXC, which was derived from rat tumor (ATCC CCL 165). XC cells were firstseeded in a roller bottle (Polystyrene, area surface=850 Cm2, Corning)in Dulbecco's Modification of Eagle's Medium (DMEM) containingpenicillin, streptomycin, C02, non-essential amino acids and 10% Fetalbovine serum (FBS) for 4 days. The conditioned medium was collected andclarified by centrifugation.

2. Assays

(a) Cell Death (Apoptosis) Assay

Prostate cancer cell line LNCAP was routinely used for the isolation ofApogen P-1 and Apogen P-2, whereas leukemia cell line HL-60 was used forthe isolation of Apogen L. The methods of assays were as follows: LNCAPor HL-60 (1,000 cells) was seeded in 10 micro-liters RPMI containing 15%or 20% Fetal bovine serum, penicillin and streptomycin at 37 degrees, 5%C02 in Microtray plates (25 μl wells, Robbins Scientific Corp.). Thetested sample (10 μl) was added 3-4 hours after cells were seeded. Afterincubation of the tested sample with cells for 15 hours, twomicro-liters of Hoechst dye (0.03 ng/ml in PBS) was added. Two hourslater, cells that were stained with Hoechst dye were examined underfluorescence microscope. The nuclei of apoptotic cells showed DNAcondensation and fragmentation, which are easily identified by Hoechstdye staining. The percentage of apoptotic cells was calculated by thefollowing equation:% Apoptotic cells=(Number of cells with DNA condensation andfragmentation)/(Total cell number)(b) Cell Repelling Assay

There are two reasons that Hep G2 cells are chosen for the study of cellrepelling activity. First, Hep G2 cells are not sensitive to Apogen P-1in inducing apoptosis. Secondly, the cell size of Hep G2 cell is about3-4 times as big as the pore size of the membrane on the TranswellInsert, which is a good cell size for cell migration/invasion study. Atissue culture device called Transwell Insert purchased from Costar(Cambridge, Mass.) was used to discover the chemorepellent activity ofApogen P-1b. This device, which has been widely used for the studies ofcell migration/invasion, contains an upper chamber and a lower chamber.Between these two chambers is a polyester microporous membrane with 3.0μm pore size, which allows cells to migrate through the membrane. Testedcells were grown on the upper chamber, and the tested compound wasplaced in the lower chamber. If this tested compound is achemoattractant, we should see more cells migrate through membrane thanthe control sample.

In our experiments, Hep G2 (100,000 cells) cells, which have a cell size3-4 times as big as the membrane pore size were grown in the upperchamber (Minimum Essential Medium Eagle containing 10 % FBS, PS andnonessential amino acid, 0.1 ml) for 2 hours, and then the partiallypurified Apogen-1b (30 μl) isolated by ammonium sulfate precipitationand Q2 HPLC chromatography as described above was placed in the lowerchamber which contains 0.6 ml of the same growth medium for Hep G2cells. After 15 hours, cells that have migrated through the membranewere collected by treating the membrane with 0.2 ml of trypsin solutionfor 30 min. Cells in ten micro-liters of the trypsin solution werecounted in a hemacytometer.

3. Protein Isolation

A. Isolation of Apogen P-1

Step 1: Ammonium Sulfate Precipitation

Apogen P-1 was precipitated by 80% saturated of ammonium sulfate byadding 561 g of ammonium sulfate per liter of XC conditioned medium. Thepellet was collected by centrifugation, and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4). After removal of ammonium sulfate bydialysis, the dissolved proteins were separated by a Q2 HPLC column.

Step 2: Q2 HPLC Chromatography

The dissolved proteins isolated by ammonium sulfate precipitation wereconcentrated and loaded onto a Q2 column (Bio Rad) which was furtherdeveloped by a linear gradient constructed by buffer A (10 mM Tris-HCl,pH 7.4) and buffer B (10 mM Tris-HCl, pH 7.4, 0.55 M NaCl) usingBioRad's BioLogic HPLC system. The linear gradient was constructed byincreasing buffer B from 0% to 100% in buffer A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% buffer B for 5 min. The Apogen P-1 activity was assayed by theinduction of apoptosis in LNCAP cells. We found that there are threeactivity peaks across the chromatogram profile. Fractions 5 to 7 cause70% cell death; fractions 8-10 cause 65% cell death; and fractions 11-14caused 90% cell death in 18 hr. We collected fractions 5-7 and named itApogen P-1a; fractions 8-10 was named Apogen P-1b; and fractions 11-14was named Apogen P-1c. These three Apogen P-1's were further purified bya reverse phase column.

Step 3: Reverse Phase Chromatography.

Apogen P-1a, Apogen P-1b and Apogen P-1c were separately concentrated to1.5 ml. One ml of methanol containing 0.05% Trifluoracetic acid wasadded. In each sample, a large amount of protein was precipitated bythis treatment, whereas the apoptosis inducing activity remained in thesupernatant. The supernatant was then applied to a reverse phase RP-4column (Micra Scientific Inc) and developed by a linear gradientconstructed by solution A (H20, 0.05% TFA) and solution B Methanol,0.05% TFA). The linear gradient was constructed by increasing solution Bfrom 0% to 100% in solution A within 10 min., 20 milliliter elutionvolume, and thereafter the column was eluted with 100% solution B for 5min.

Step 4: Preparative Electrophoresis

Apogen 1c isolated by anion exchange chromatography was purified by bothReverse Phase Chromatography (step 3) and Preparative Electrophoresis bya MiniPrep Gel electrophoresis (Bio-Rad). In the reverse phasechromatogram of Apogen P-1a, fractions 12-13 have activity inducing 80%cell death in LNCAP cells at 10 hr.

In the reverse phase chromatogram of Apogen P-1b, fractions 14 and 15have activity inducing 45% cell death in LNCAP cells at 18 hr.

In the reverse phase chromatogram of Apogen P-1c, fraction No. 5 hasactivity inducing 52% cell death in LNCAP cells at 18 hr.

The purity of the isolated Apogen P-1a, Apogen P-1b and Apogen P-1c werechecked with SDS-polyacrylamide gel electrophoresis stained with silverstaining.

-   (1) Apogen P-1a: A protein band with molecular weight of 70 KD was    obtained. This result suggests the nearly successful purification of    Apogen P-1a, which has a molecular weight of 70 KD on SDS-PAGE.-   (2) Apogen P-1b: A single faint protein band with molecular weight    of 55 KD was obtained. This result suggests the successful    purification of Apogen P-1b, which has a molecular weight of 55 KD    on SDS-PAGE.-   (3) Apogen P-1c: The purification of Apogen 1c by Reverse Phase    chromatography leads to the Isolation of a 70 KD protein, whereas    the purification of Apogen 1c by preparative electrophoresis leads    to the purification of a 57 KD protein. A major protein band with    molecular weight of 70 KD was obtained by Reverse Phase    chromatography. A 57 KD protein, on the other hand, was isolated by    preparative electrophoresis.    B. Isolation of Apogen P-2

The Apogen P-2 present in C3H1OT1/2 conditioned medium was isolated bythe following steps:

Step 1: Ammonium Sulfate Precipitation.

Apogen P-2 was precipitated by ammonium sulfate (80% saturated) byadding 561 g of ammonium sulfate per liter of conditioned medium. Thepellet was collected by centrifugation, and the proteins were dissolvedin 10 mM Tris-HCl (pH 7.4).

Step 2: Hydroxylapatite Treatment.

After removal of ammonium sulfate by dialysis in 10 mM Tris-HCl (pH7.5), the dissolved proteins were incubated with hydroxylapatite gel(Bio-Gel HTP gel, Bio-Rad) for 1 hr. After removing HTP gel bycentrifugation, the activity inducing apoptosis in LNCAP cells was foundto be present in the supernatant, which was then further treated withHeparin agarose gel.

Step 3: Heparin Agarose Treatment.

The supernatant from step 2 was further incubated with Heparin agarose(Sigma) for 1 hr. After removing HTP gel by centrifugation, the activityinducing apoptosis in LNCAP cells was found to be present in thesupernatant.

Step 4: Reverse Phase Chromatography.

Apogen P-2 presents in the supernatant of Heparin agarose in step 3 wasfurther purified by reverse phase chromatography. Apogen P-2 wasconcentrated to 1 ml. One milliliter of methanol containing 0.05%trifluoacetic acid was added. Large amounts of proteins wereprecipitated by this treatment, whereas the apoptosis inducing activity(P-2) remained in the supernatant. The supernatant was then applied to areverse phase RP-4 column (Micra Scientific Inc.) and developed by alinear gradient constructed by solution A (H20, 0.05% TFA) and solutionB Methanol (0.05% TFA). The linear gradient was constructed byincreasing solution B from 0% to 100% in solution A within 10 min (20milliliter elution volume) and thereafter the column was eluted with100% solution B for 5 min. In the reverse phase chromatogram of ApogenP-2, fractions 12-14 have activity inducing 80% cell death in LNCAPcells at 12 hr. The purity of the isolated Apogen P-2 was checked withSDS-polyacrylamide gel electrophoresis stained with silver staining. Asingle protein band with molecular weight of 65 Kd was obtained.

C. Isolation of Apogen L

The Apogen L present in the conditioned medium was isolated by thefollowing steps:

Step 1: DE52 Absorption

The conditioned medium was incubated with the anion exchanger, DE 52(Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubation mixturewas centrifuged, and DE 5 2, which binds Apogen L, was collected andwashed with 10 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. Apogen L wasthen eluted from DE 5 2 cellulose by 10 mM Tris-HCl (pH 7.5) containing0.5 M NaCl.

Step 2: Heparin Agarose Absorption

Apogen L isolated as described in step 1 was further absorbed by Heparinagarose (Sigma) by incubating Apogen L with Heparin agarose for 1 hr.Heparin agarose was collected by centrifugation and was washed with 10mM Tris-HCl (pH 7.5). Apogen L absorbed in Heparin agarose was theneluted by 2 M NaCl.

Step 3: Q2 HPLC Chromatography

Apogen L isolated as described above was concentrated and loaded onto aQ2 column (Bio Rad) which was further developed by a linear gradientconstructed by buffer A (10 mM Tris-HCl, pH 7.4) and buffer B (10 mM 22Tris-HCl, pH 7.4, 0.5 M NaCl) using Bio-Rad's BioLogic HPLC system. Thelinear gradient was constructed by increasing buffer B from 0% to 100%in buffer A in 10 min. The purity of the isolated Apogen L was checkedwith SDS-polyacrylamide gel electrophoresis stained with silverstaining. A single protein band having activity with a molecular weightof approximately 55 Kd was obtained.

4. Isolation of Bovine Fetuin as a Component of Protein P-2 and theApoptotic Effect Thereof in Tumor Cell Lines.

The observation that Apogen P-1a, P-1b, P-1c, P-2 and L were isolatedfrom embryonic cell lines led us to speculate that newborn or embryonictissue may secrete “Apogen,” which may selectively induce apoptosis intumor cell lines. Thus, due to this speculation, a protein named“Fetuin” has raised our attention for the following reasons: (1) Fetuinis mainly a fetal protein, in the sense that the highest concentrationsare found in serum and body fluids of embryos and fetuses. For example,the concentration of fetuin in bovine serum drastically decreases,probably within a few days after birth, to 1-2% of the fetal level.(Yang, et al., Biochim. Biophy. Acta. 1130, 149-156 1992). (2) Ahistochemical study has shown that fetuin may control tissue remodellingand physiological cell death during embryonic development. (Von Bulow,et al., Histochemistry 99:13-22, 1993). This result raises thepossibility that fetuin may contain activity inducing cell death(apoptosis).

Additionally, a protein with an amino acid sequence identical to Fetuinwas isolated from the preparation of Apogen P-2. Thus, the compositionof Apogen P-2 consists at least in part of fetuin.

We therefore prepare and obtain fetuin and test fetuin in our apoptosisassay. Interestingly, we found that only bovine fetuin that is preparedby a special method is able to induce apoptosis in tumor cell lines. Thecommercial fetuin that is prepared by ammonium sulfate precipitation andEDTA treatment was found to contain a very low activity in inducingapoptosis in tumor cells.

4A. Preparation of Bovine Fetuin.

Bovine fetuin was prepared by the modified Spiro method (Spiro R. G.,Journal of Biological Chemistry 235, 10: 2860, 1960) according to thefollowing steps:

1. One hundred milliliters of Fetal Bovine Serum (FBS).

2. Add two hundred milliliters of 0.05 M Zinc Acetate containing 30%(V/V) ethanol, adjust to pH 6.4 by 1M NH4OH-NH4Cl, let stand 15 hours at−5° C.

3. Collect the supernatant by centrifugation, add 1.0 M Barium Acetateand 95% ethanol to give 0.03 M Barium Acetate, 25% ethanol. Let stand 2hours at −5° C.

4. Collect the supernatant by centrifugation, add 95% ethanol to give40% ethanol. Let stand 15 hours at −10° C.

5. Collect the precipitate. Dissolve the pellet by phosphate buffersaline.

The purified fetuin showed a single protein band with apparent molecularweight of 63 Kd on SDS-PAGE.

4B. Induction of Apoptosis in Tumor Cell Lines Using Bovine Fetuin.

Fetuin purified from fetal bovine serum by the procedure described abovewas dissolved in phosphate buffer saline (PBS). The free Zinc Acetateand Barium Acetate were removed by repetitive concentration. Fetuin wastested in LNCaP and HL-60 cells. LNCaP or HL-60 (1,000 cells) was seeded10 micro-liters RPMI containing 15% or 20% Fetal bovine serum,penicillin and streptomycin at 37 degree, 5% CO2 in microtray plates (25μl wells, Robbins Scientific Corp.). Fetuin (in 10 μl PBS) atconcentration of 100 ng/ml was added 3-4 hours after cells were seeded.After incubation of the tested sample with cells for 15 hours, twomicro-liters of Hoechst dye (0.03 ng/ml in PBS) was added. Two hourslater, cells that were stained with Hoechst dye were examined underfluorescence microscope. The nuclei of apoptotic cells showed DNAcondensation and fragmentation, which can be easily identified byHoechst dye staining. The percentage of apoptotic cells was calculatedby the following equation:% Apoptotic cells=(Number of cells with DNA condensation andfragmentation)/(Total cell number)

The nuclei of the LNCaP cells that have been incubated with controlsample (PBS) are normal and healthy. However, the nuclei of the LNCaPcells that have been incubated with fetuin (100 ng/ml in PBS) show thecharacteristics of apoptosis. First, the cells in the presence of fetuinshowed the condensation of nucleus, demonstrated by the more intensefluorescent light compared with the control nucleus. Secondly, thenucleus condensation is accompanied by the fragmentation of DNA,demonstrated by the breakage of nucleus.

As the condensation of the nucleus and the DNA fragmentation are the twomorphological characteristics of cells under apoptosis, these resultssuggest that fetuin contains an activity inducing apoptosis in LNCaPcells. The nuclei of the HL-60 cells that have been incubated withcontrol buffer (PBS) are normal and healthy. However, the nuclei of theHL-60 cells that have been incubated with fetuin show thecharacteristics of apoptosis. Fetuin causes the condensation of nucleus,demonstrated by the more intense fluorescent light compared with thecontrol nucleus. Secondly, the nucleus condensation is accompanied bythe fragmentation of DNA, demonstrated by the breakage of the nucleus.As we have mentioned above, the nucleus condensation and DNAfragmentation are the two morphological characteristics of cells underapoptosis. These results suggest that fetuin contains an activityinducing apoptosis in HL-60 cells.

4C. Bovine Fetuin Selectively Induces Apoptosis in Cancer Cells WithoutHaving an Effect on Normal Cell Lines.

We compared the effect of fetuin on the induction of apoptosis invarious cell lines. At a concentration of 50 μg/ml, fetuin prepared asdescribed above strongly induced apoptosis in tumor cell lines such as:LNCaP (prostate cancer), PC-3 (prostate cancer), HL-60 (leukemia), MCF-7(breast cancer), Colo 205 (colon cancer), Calu-1 (lung cancer). Normallung fibroblast (CCD 39 Lu), on the other hand, is not affected byfetuin.

Fetuin was found to be inactive in inducing apoptosis in CCD 39 Lu cells(normal lung fibroblast) at the concentration (25 μg/ml) that highlyinduced apoptosis in LNCaP (prostate cancer) or HL-60 cells (leukemia).Fetuin (25 μg/ml) prepared as described above was incubated with CCD 39Lu cells grown in MEM in a microtray plate for 15 hours. The CCD 39 Lucells remained morphologically unchanged in the presence of fetuin. Atthis concentration (25 μg/ml) of fetuin and in the presence of fetuin,less MCF-7 cells remaining, due to cell death and cell shrinkage, wasobserved.

4D. Only Fetuin Prepared by the Method Described Above is Able to InduceApoptosis in Tumor Cell Lines.

We found that fetuin purchased from Sigma has a very low activity ininducing apoptosis in LNCaP cells. However, fetuin (25 μg/ml) preparedin our laboratory by the method described in Section 4A above inducesapoptosis in LNCaP cells by up to 90% in 4 hours. For the fetuinpurchased from Sigma, apoptosis-inducing activity was observed only at avery high concentration (>250 μg/ml) and at long incubation time (2days). We estimated that the activity of fetuin prepared in ourlaboratory is more than fifty thousand folds higher than that of fetuinprepared by other methods.

In the years of research following the original findings, I haveobserved that fetuin from Sigma induces apoptosis at a very highconcentration and at a long incubation time (2 days). It isconservatively estimated that the fetuin as prepared in Section 4A hasmore than one hundred times greater apoptotic activity than fetuinprepared by other methods. While this is not as dramatic as the 50,000times increase as originally reported, it still represents a significantapoptotic advantage over previously available fetuin in terms ofincubation time and LD₅₀ values.

We have examined the preparation method for Sigma's fetuin and foundthat these fetuins are prepared by methods including ammonium sulfateprecipitation and EDTA treatment. Both treatments may cause thedeprivation of the Zinc ion from the protein, which may cause theirreversible loss of the protein activity.

4E. Effect of Fetal Fetuin on Leukemia Cells In Vivo.

Our previous data demonstrated that fetuin induced apoptosis in cancercells in vitro. The data provided below shows that the in vivo testingof fetuin in mice having leukemia was successful. The results show thatfetuin has an anti-leukemia effect in mice. FIG. 1 shows the increase insurvival of leukemia-bearing mice treated with fetal fetuin.

METHOD

Forty DBA/2 female mice (17-20 grams; Simonsen Laboratories, Inc.,Gilroy, Calif.) kept on a standard diet and water ad libitum wereinoculated with tumor cell line P388D1 (ATCC cell line number CCL46).The mice were randomly segregated into groups of ten (10). Zinc-chargedfetal fetuin (10 mg/ml) were intra-peritoneally injected into group I at0.002 ml/mouse, group II at 0.02 ml/mouse and group III at 0.2 ml/mouse.Group IV was the control group, which was injected with 0.5 ml of salinesolution. The injections were continued for 10 days. Mortalities wererecorded for 60 days. The results were expressed as the percentageincrease in life span (ILS):ILS=(100×Median Life Span Treated−Median Life Span Controlled)/(LifeSpan Controlled)

FIG. 1 shows that while 100% of the untreated leukemia-bearing mice weredead after 24 days, 80% of the mice treated with a high dose of fetuin,namely 100 mg/Kg of fetal fetuin, survived more than 58 days. This invivo experiment demonstrates that mice bearing leukemia that are treatedwith fetal fetuin have an increased life span of 141%.

4F. Method of Preparing Supercharged Zinc Fetuin:

The method to prepare fetuin with zinc has been refined and improved. Asstated above, fetuin prepared by the method as described in Section 4Aabove is able to induce apoptosis in tumor cell lines. However,commercial fetuin such as from Sigma is found to have a very lowactivity in inducing apoptosis in tumor cells and in inducing apoptosisin LNCaP cells. For fetuin from Sigma, apoptosis inducing activity wasobserved only at a very high concentration (>250 μg/ml) and at a longincubation time (2 days), whereas fetuin (25 μg/ml) as prepared inSection 4A above induced apoptosis in LNCaP cells by up to 90% in 4hours. It was estimated that the fetuin as prepared as described inSection 4A is more than fifty thousand times higher than that fetuinprepared by other methods. The radically different results suggest afundamental difference in the chemical composition of commerciallyavailable fetuin and the fetuin prepared in accordance with theprocedure in Section 4A.

In the years of research following the original findings, I haveobserved that fetuin from Sigma induces apoptosis at a very highconcentration (>>5 mM) and at a long incubation time (2 days), whereasfetuin (approximately 50 μM) as prepared in Section 4A above inducedapoptosis in LNCaP cells by up to 90% in 4 hours. It is conservativelyestimated that the fetuin as prepared in Section 4A has more than onehundred times greater apoptotic activity than fetuin prepared by othermethods. While this is not as dramatic as the 50,000 times increase asoriginally reported, it still represents a significant apoptoticadvantage over previously available fetuin in terms of incubation timeand LD₅₀ values.

After looking at the methods of preparing commercially available fetuin,it was found that ammonium sulfate precipitation and EDTA treatment wasused in preparing fetuin. It was speculated that this ammonium sulfateprecipitation and EDTA treatment might cause the deprivation of the ionsfrom the protein to cause irreversible loss of the protein activity.However, it was not known whether it was the loss of zinc alone, or incombination with the loss of another ion(s), that caused the decreasedapoptotic activity in commercially available fetuin.

While it was determined that Fetuin-Ca is inactive in inducing apoptosis(data not shown), and barium occurs in only trace amounts, to determinewhich ion, or combination of ions, were most effective in increasing theapoptotic ability of fetuin, fetuin as prepared in Section 4A above wastreated with a chelating agent, such as EDTA, to strip all the inorganicions, including zinc, calcium, and barium from the protein. Afterremoving these inorganic ions, the “naked” fetuin was treated orincubated with 0.5 M Zinc Acetate to reload or to bind the fetuin withzinc only. The results of this refinement process are shown in examplesin FIGS. 2-3 in determining the LD₅₀ value. FIGS. 2-3 show that theimproved preparation of Fetuin-Zn or “supercharged zinc fetuin” enhancesfetuin's ability to induce apoptosis in cancer cells by three to fourtimes as compared with the original fetuin bound with zinc as preparedin Part 4A. It is hypothesized that the fetuin previously bound up withcalcium and barium created an inactive form of the protein. By strippingout all ions and replacing them with zinc, inactive fetuin moleculeswere converted to active form, thereby explaining the dramatic increasein apoptotic activity. Such supercharged zinc fetuin is a valuable stepforward in the fight against cancer.

In one preferred embodiment of this preparation process:

1. Incubation Mixture: 700 μg of fetuin (0.2 ml; as prepared by themethod as described above in Section 4A) was incubated with 0.5 ml of0.1 M EDTA for approximately one (1) hour.

2. Concentration: Add 1.5 ml of saline solution to this incubationmixture and concentrate to near dryness using a molecular or moleculesieve and centrifugal force. Repeat this procedure four (4) times, sothat most of the inorganic ions are removed. This “naked” fetuin will beretained on the top of the filter (molecular sieve).

3. Incubate the “naked” fetuin (0.2 ml) with 0.5 ml of 0.5 M ZincAcetate for approximately three (3) hours.

4. Remove the free Zinc Acetate using the combination of the salinesolution, the molecular sieve, and centrifugal force as described inStep 2 above.

5. A Specific Peptide Fragment From Fetuin-Zinc That Causes Apoptosis inCancer Cells.

a. Preparation of Fetuin Fragment:

As described above in Section 4F (Method of Preparing Supercharged ZincFetuin), zinc charged fetuin or supercharged zinc fetuin was prepared bypre-treatment of fetal bovine fetuin with a chelating agent (EDTA) toremove the inorganic ions, including zinc, calcium, and barium ions,from the fetuin. The resulting stripped fetuin was incubated with 0.5 MZinc Acetate in order to “supercharge” or load the fetuin with zinc.Three hundred (300) micrograms of the zinc charged fetuin was dissolvedin a 50 μl saline solution and then dried in a tube under a vacuum. Itis hypothesized that this drying step breaks apart the zinc chargedfetuin into peptide fragments.

The dried fragments (of zinc charged fetuin) of were reconstituted in 50μl water. This fragment solution was passed through a molecular sievemembrane having a molecular weight cut-off of 10,000 daltons. Theresulting filtrate of fragments was collected and tested on cells in anapoptosis assay. As shown in FIG. 5, when LNCaP cells are incubated withthe zinc charged fetuin fragment filtrate for six (6) hours, the LNCaPcells detach and die. Compared with the control (LNCaP with no filtrate)as shown in FIG. 4, FIG. 5 shows that incubation of the prostate cancercells with the zinc charged fetuin filtrate causes apoptosis of thecancer cells. FIG. 7 shows that the zinc charged fetuin filtrate treatedLNCaP cells also exhibit membrane “blebbing,” which is a characteristictypical of cells undergoing apoptosis. FIG. 6, which shows LNCaP cellswithout the zinc charged fetuin filtrate, lacks this membrane “blebbing”and characteristic of apoptosis.

b. Is Protease Sensitive.

Additionally, the apoptosis-inducing activity of FPF 300-09 was found tobe protease sensitive. Incubation of the FPF 300-09 filtrate withproteinase K completely removed the apoptosis-inducing activity.Proteinase K is an enzyme that cleaves peptide bonds; chelating agentssuch as citrate and EDTA have no effect on the enzyme activity ofproteinase K.

After preparing “supercharged” zinc fetuin or zinc charged fetuin asstated in Section 4F above, the resulting composition was dried in atube and under a vacuum. This dried supercharged zinc fetuin wasreconstituted in 50 μl of water. This solution was filtered through amolecular sieve membrane (Centricon 10 tube with a molecular weightcut-off: 10,000 daltons). The filtrate was collected and treated with 5μl (1 unit/μl) proteinase K for three (3) hours at 37° C. Aftertreatment with proteinase K, the treated filtrate was filtered through amolecular sieve membrane (Centricon 10 tube) in order to remove theproteinase K. The proteinase K was retained by the membrane and thetreated filtrate passed through the membrane.

To test the effect of a protease on the apoptotic activity of thefiltrate, the filtrate treated with proteinase K was tested on cancercells. These results were compared to the filtrate that was not treatedwith proteinase K.

The effect of proteinase K on the apoptotic ability of the Fetuin-Zincfragments is summarized in FIG. 8. Experiments 1 and 2 were conductedwith one set of zinc charged fetuin filtrate, and Experiment 3 wasconducted with another set of zinc charged fetuin filtrate. FIG. 8 showsthat incubation with a protease seems to inactivate the apoptotic effectof the zinc charged fetuin fragment. Because a protease, such asproteinase K, cleaves peptide bonds, the test results of FIG. 8 stronglysuggest that a peptide or a protein of fetuin is responsible for theinduction of apoptosis in cancer cells.

c. The Filtrate Contains Two Major Peptides Derived From Fetuin.

The dried and reconstituted filtrate was found to contain peptidefragments. The amino acid sequence analysis revealed two major peptidefragments in the filtrate:

(1) His Thr Phe Ser Gly Val Ala Ser Val Glu (amino acid no. 300-309; SEQID NO:1) and

(2) Ser Ala Ser Gly Glu Ala Phe His (amino acid no. 310-317; SEQ IDNO:2) of fetuin.

To identify which of these peptide fragments are responsible for theapoptosis-inducing activity, the two fragments (His Thr Phe Ser Gly ValAla Ser Val Glu (amino acid no. 300-309; referred to herein as FetuinPeptide Fragment (FPF 300-09); SEQ ID NO:1) and Ser Ala Ser Gly Glu AlaPhe His (amino acid no. 310-317; SEQ ID NO:2) of the full-length fetuinmolecule) were chemically synthesized. Upon in vitro testing of thesechemically synthesized peptide fragments, FPF 300-09 was shown to havethe greater apoptotic activity. LNCaP (prostate cancer cells) wereincubated with FPF 300-09. In FIG. 10, chemically synthesized FPF 300-09caused membrane “blebbing” in LNCaP cells after three (3) hours ofincubation. Incubation of the peptide fragment (amino acid no. 310-317)or the peptide fragment (amino acid no. 300-307) with LNCaP cells didnot show any apoptotic activity or membrane “blebbing.” FIG. 9 shows thecontrol of LNCaP cells without FPF 300-09. These results suggest thatthe peptide fragment that induced apoptosis and that was present in thefiltrate corresponds to amino acid no. 300-309 of the full-lengthfetuin.

6. Characterization of FPF 300-09.

a. FPF 300-09 Selectively Induced Apoptosis in Cancer Cells But Not inNormal Cells.

Previously, it was found that fetuin and zinc charged fetuin inducedapoptosis in various cancer cells without affecting certain normalcells. To test whether FPF 300-09 derived from fetuin retains thisselectivity in inducing apoptosis in cancer cells only and not affectingnormal cells, various concentrations of FPF 300-09 were tested on HT-29(colon cancer), CCD-18 Co (normal colon), and LNCaP (prostate cancer)cells. As shown in FIG. 11, FPF 300-09 induced apoptosis in HT-29 andLNCaP cells without affecting CCD-18 Co cells. These results suggestthat the fragment was similar to fetuin and zinc charged fetuin inselectively inducing apoptosis in cancer cells and not in normal cells.

b. FPF 300-09 Rapidly Induced Apoptosis in HT-29 (Colon Cancer) Cells.

FIG. 12 shows the effect of time on the induction of apoptosis by FPF300-09. At four (4) hours, FPF 300-09 at a concentration of 0.4 μMinduced apoptosis in 52% of the HT-29 colon cancer cells. At six (6)hours, almost all of the HT-29 cells were induced to apoptosis. Evenwith a little more incubation time (from 4 to 6 hours), the apoptoticability of FPF 300-09 rapidly increases within this short period oftime.

c. The Peptide Fragment Derived From Fetuin is More Potent Than Fetuinor Zinc Charged Fetuin in Inducing Apoptosis.

Previously, the LD₅₀ (dosage for the induction of 50% cell death) forfull-length zinc charged fetuin in LNCaP cells was determined to be 3-10μM. However, the LD₅₀ for FPF 300-09 was determined to be 0.3-0.4 μM.Hence, with a much smaller LD₅₀ value, a much smaller amount of the FPF300-09 was required to induce the same amount of cancer cells than isrequired with full-length zinc charged fetuin. Further, FPF 300-09 ismore potent in inducing apoptosis than its parent molecule (FIG. 13).

Taking into consideration the previous estimates:

-   (1) Fetuin as prepared in Section 4A above is approximately 100    times more powerful than fetuin prepared from other methods;-   (2) “Supercharged” Zinc Fetuin (zinc charged fetuin) is    approximately three to four times more powerful than the fetuin as    prepared in Section 4A (see FIGS. 2-3); and-   (3) FPF 300-09 is approximately eight to ten times more powerful    than “Supercharged” Zinc Fetuin (see FIG. 13),    it is estimated that FPF 300-09 is approximately several thousand    times more powerful than fetuin prepared from other methods.    7. Other Sequences of Fetuin From Other Sources.

In addition, peptide sequences were determined from other animal sera,including pig, sheep and mice. K. M. Dziegielewska, et. al., Fetuin,16-17,(R. G. Landes Co. 1995). These peptide sequences have a similarityof 60-90% with the fetuin isolated from bovine serum. The presentapplication instructs that these similar fetuin peptide sequences willalso have valuable apoptotic activity. The peptide sequences for FPF300-09 for other species are:

Human (His Thr Phe Met Gly Val Val Ser Leu Gly; SEQ ID NO:3);

Pig (His Ser Phe Ser Gly Val Ala Ser Val Glu; SEQ ID NO:4); and

Mouse (His Ala Phe Ser Pro Val Ala Ser Val Glu; SEQ ID NO:5). Id.

Sheep and rat species have the fetuin peptide sequence identical to thecow sequence (SEQ ID NO: 1). These analogs are also claimed as beingtaught by the current application.

DISCUSSION

This invention describes the methods for the isolation of five proteins(Apogen P-1a, Apogen P-1b, Apogen P-1c, Apogen P-2 and Apogen L) thatare able to induce apoptosis in prostate cancer cells (Apogen P-1's), inprostate cancer cells and breast cancer cells (Apogen P-2), and leukemiaand breast cancer cells (Apogen L), as well as the identification offetuin as a component of Apogen P-2. The following evidence leads us tobelieve that these apoptosis-inducing proteins are novel and that theyhave never been found before: Tumor Necrosis Factor (TNF), TransformingGrowth Factor (TGF-Beta), Fas ligand and TRAIL are the proteins reportedto induce apoptosis in certain cell lines. (Lin, J. K. et al., CancerResearch 52:385, 1992. Kawakawi, et al., J. of Cellular Physiology138:1, 1989; Wiley, S. R. et al., Immunity 3:673, 1995; Krammer, et al.“Apoptosis in the APO-1 System”, Apoptosis: The molecular Basis of CellDeath, Cold Spring Harbor Laboratory Press p. 87, 1991). Evidencesuggests that these five proteins are different from any of these knownproteins inducing apoptosis as described below:

(1) The activities are different. In our assays, TNF and TGF inducedapoptosis in liver cancer cells without effect in prostate cancer(LNCAP) cells even if a very high dose (100 ng/ml) is used, whereasApogen P-1's and Apogen P-2 induced apoptosis in prostate cancer ratherthan in cancer liver cells.

(2) TRAIL and Fas are membrane bound proteins, (Wiley, S. R. et al.Immunity 3:673, 1995; Krammer, et al., “Apoptosis in the APO-1 System”,Apoptosis: The molecular Basis of Cell Death, Cold Spring HarborLaboratory Press, p. 87, 1991) whereas the Apogen P-1a, Apogen P-1b,Apogen P-1c, Apogen P-2 and Apogen L are all soluble (non-membranebound) proteins.

(3) The molecular weights of TNF, TGF and Fas ligand TRAIL are around17-40 Kd (TNF=17 KD, TGF=24 KD, TRAIL=32 KD, Fas ligand=43 KD) (McGrath,M. H. Clinics in Plastic surgery 17:421, 1993; Wiley, S. R. et al.,Immunity 3:673, 1995; Krammer, et al., “Apoptosis in the APO-1 System”,Apoptosis: The molecular Basis of Cell Death, Cold Spring HarborLaboratory Press, p. 87, 1991) whereas the molecular weights of ApogenP-1a, Apogen P-1b, Apogen P-1c, Apogen P-2 and Apogen L are between55-70 Kd.

1. An isolated composition for inducing apoptosis in cancer cellsconsisting of the amino acid sequence His Thr Phe Met Gly Val Val SerLeu Gly (SEQ ID NO: 3) and zinc.
 2. The composition of claim 1 whereinsaid cancer cells are prostate cancer cells.
 3. The composition of claim1 wherein said cancer cells are colon cancer cells.